Role of cofilin in epidermal growth factor-stimulated actin polymerization and lamellipod protrusion

Stimulation of metastatic MTLn3 cells with epidermal growth factor (EGF) causes a rapid and transient increase in actin nucleation activity resulting from the appearance of free barbed ends at the extreme leading edge of extending lamellipods. To investigate the role of cofilin in EGF-stimulated actin polymerization and lamellipod extension in MTLn3 cells, we examined in detail the temporal and spatial distribution of cofilin relative to free barbed ends and characterized the actin dynamics by measuring the changes in the number of actin filaments. EGF stimulation triggers a transient increase in cofilin in the leading edge near the membrane, which is precisely cotemporal with the appearance of free barbed ends there. A deoxyribonuclease I binding assay shows that the number of filaments per cell increases by 1.5-fold after EGF stimulation. Detection of pointed ends in situ using deoxyribonuclease I binding demonstrates that this increase in the number of pointed ends is confined to the leading edge compartment, and does not occur within stress fibers or in the general cytoplasm. Using a light microscope severing assay, cofilin's severing activity was observed directly in cell extracts and shown to be activated after stimulation of the cells with EGF. Microinjection of function-blocking antibodies against cofilin inhibits the appearance of free barbed ends at the leading edge and lamellipod protrusion after EGF stimulation. These results support a model in which EGF stimulation recruits cofilin to the leading edge where its severing activity is activated, leading to the generation of short actin filaments with free barbed ends that participate in the nucleation of actin polymerization.     

Measurement of barbed ends, actin polymerization, and motility in live carcinoma cells after growth factor stimulation

Motility is associated with the ability to extend F-actin-rich protrusions and depends on free barbed ends as new actin polymerization sites. To understand the function and regulation of different proteins involved in the process of generating barbed ends, e.g., cofilin and Arp2/3, fixed cell approaches have been used to determine the relative barbed end concentration in cells. The major disadvantages of these approaches are permeabilization and fixation of cells. In this work, we describe a new live-cell time-lapse microscopy assay to determine the increase of barbed ends after cell stimulation that does not use permeabilization and provides a better time resolution. We established a metastatic carcinoma cell line (MTLn3) stably expressing GFP-beta-actin at physiological levels. Stimulation of MTLn3 cells with epidermal growth factor (EGF) causes rapid and transient lamellipod protrusion along with an increase in actin polymerization at the leading edge, which can be followed in live cell experiments. By measuring the increase of F-actin at the leading edge vs. time, we were able to determine the relative increase of barbed ends after stimulation with a high temporal resolution. The F-actin as well as the barbed end concentration agrees well with published data for this cell line. Using this newly developed assay, a decrease in lamellipod extension and a large reduction of barbed ends was documented after microinjecting an anti-cofilin function blocking antibody. This assay has a high potential for applications where rapid changes in the dynamic filament population are to be measured.     

Phospholipase C and cofilin are required for carcinoma cell directionality in response to EGF stimulation

The epidermal growth factor (EGF)-induced increase in free barbed ends, resulting in actin polymerization at the leading edge of the lamellipodium in carcinoma cells, occurs as two transients: an early one at 1 min and a late one at 3 min. Our results reveal that phospholipase (PLC) is required for triggering the early barbed end transient. Phosphoinositide-3 kinase selectively regulates the late barbed end transient. Inhibition of PLC inhibits cofilin activity in cells during the early transient, delays the initiation of protrusions, and inhibits the ability of cells to sense a gradient of EGF. Suppression of cofilin, using either small interfering RNA silencing or function-blocking antibodies, selectively inhibits the early transient. Therefore, our results demonstrate that the early PLC and cofilin-dependent barbed end transient is required for the initiation of protrusions and is involved in setting the direction of cell movement in response to EGF.     

Relationship between Arp2/3 complex and the barbed ends of actin filaments at the leading edge of carcinoma cells after epidermal growth factor stimulation

Using both light and high resolution electron microscopy, we analyzed the spatial and temporal relationships between the Arp2/3 complex and the nucleation activity that is required for lamellipod extension in mammary carcinoma cells after epidermal growth factor stimulation. A rapid two- to fourfold increase in filament barbed end number occurs transiently after stimulation and remains confined almost exclusively to the extreme outer edge of the extending lamellipod (within 100-200 nm of the plasma membrane). This is accompanied by an increase in filament density at the leading edge and a general decrease in filament length, with a specific loss of long filaments. Concomitantly, the Arp2/3 complex is recruited with a 1.5-fold increase throughout the entire cortical filament network extending 1-1.5 microm in depth from the membrane at the leading edge. The recruitment of the Arp2/3 complex at the membrane of the extending lamellipod indicates that Arp2/3 may be involved in initial generation of growing filaments. However, only a small subset of the complex present in the cortical network colocalizes near free barbed ends. This suggests that the 100-200-nm submembraneous compartment at the leading edge of the extending lamellipod constitutes a special biochemical microenvironment that favors the generation and maintenance of free barbed ends, possibly through the locally active Arp2/3 complex, severing or decreasing the on-rate of capping protein. Our results are inconsistent with the hypothesis suggesting uncapping is the dominant mechanism responsible for the generation of nucleation activity. However, they support the hypothesis of an Arp2/3-mediated capture of actin oligomers that formed close to the membrane by other mechanisms such as severing. They also support pointed-end capping by the Arp2/3 complex, accounting for its wide distribution at the leading edge.     

Modeling the Synergy of Cofilin and Arp2/3 in Lamellipodial Protrusive Activity

Rapid polymerization of actin filament barbed ends generates protrusive forces at the cell edge, leading to cell migration. Two important regulators of free barbed ends, cofilin and Arp2/3, have been shown to work in synergy (net effect greater than additive). To explore this synergy, we model the dynamics of F-actin at the leading edge, motivated by data from EGF-stimulated mammary carcinoma cells. We study how synergy depends on the localized rates and relative timing of cofilin and Arp2/3 activation at the cell edge. The model incorporates diffusion of cofilin, membrane protrusion, F-actin capping, aging, and severing by cofilin and branch nucleation by Arp2/3 (but not G-actin recycling). In a well-mixed system, cofilin and Arp2/3 can each generate a large pulse of barbed ends on their own, but have little synergy; high synergy occurs only at low activation rates, when few barbed ends are produced. In the full spatially distributed model, both synergy and barbed-end production are significant over a range of activation rates. Furthermore, barbed-end production is greatest when Arp2/3 activation is delayed relative to cofilin. Our model supports a direct role for cofilin-mediated actin polymerization in stimulated cell migration, including chemotaxis and cancer invasion.     

Modeling the Synergy of Cofilin and Arp2/3 in Lamellipodial Protrusive Activity

Rapid polymerization of actin filament barbed ends generates protrusive forces at the cell edge, leading to cell migration. Two important regulators of free barbed ends, cofilin and Arp2/3, have been shown to work in synergy (net effect greater than additive). To explore this synergy, we model the dynamics of F-actin at the leading edge, motivated by data from EGF-stimulated mammary carcinoma cells. We study how synergy depends on the localized rates and relative timing of cofilin and Arp2/3 activation at the cell edge. The model incorporates diffusion of cofilin, membrane protrusion, F-actin capping, aging, and severing by cofilin and branch nucleation by Arp2/3 (but not G-actin recycling). In a well-mixed system, cofilin and Arp2/3 can each generate a large pulse of barbed ends on their own, but have little synergy; high synergy occurs only at low activation rates, when few barbed ends are produced. In the full spatially distributed model, both synergy and barbed-end production are significant over a range of activation rates. Furthermore, barbed-end production is greatest when Arp2/3 activation is delayed relative to cofilin. Our model supports a direct role for cofilin-mediated actin polymerization in stimulated cell migration, including chemotaxis and cancer invasion.     

How is actin polymerization nucleated in vivo?

Actin polymerization in vivo is dependent on free barbed ends that act as nuclei. Free barbed ends can arise in vivo by nucleation from the Arp2/3 complex, uncapping of barbed ends on pre-existing filaments or severing of filaments by cofilin. There is evidence that each mechanism operates in cells. However, different cell types use different combinations of these processes to generate barbed ends during stimulated cell motility. Here, I describe recent attempts to define the relative contributions of these three mechanisms to actin nucleation in vivo. The rapid increase in the number of barbed ends during stimulation is not due to any single mechanism. Cooperation between capping proteins, cofilin and the Arp2/3 complex is necessary for the development of protrusive force at the leading edge of the cell: uncapping and cofilin severing contributing barbed ends, whereas activity of the Arp2/3 complex is necessary, but not sufficient, for lamellipod extension. These results highlight the need for new methods that enable the direct observation of actin nucleation and so define precisely the relative contributions of the three processes to stimulated cell motility.     

A temporal model of cofilin regulation and the early peak of actin barbed ends in invasive tumor cells

Cofilin is an important regulator of actin polymerization, cell migration, and chemotaxis. Recent experimental data on mammary carcinoma cells reveal that stimulation by epidermal growth factor (EGF) generates a pool of active cofilin that results in a peak of actin filament barbed ends on the timescale of 1 min. Here, we present results of a mathematical model for the dynamics of cofilin and its transition between several pools in response to EGF stimulation. We describe the interactions of phospholipase C, membrane lipids (PIP₂), and cofilin bound to PIP₂ and to F-actin, as well as diffusible cofilin in active G-actin-monomer-bound or phosphorylated states. We consider a simplified representation in which the thin cell edge (lamellipod) and the cell interior are represented by two compartments that are linked by diffusion. We demonstrate that a high basal level of active cofilin stored by binding to PIP₂, as well as the highly enriched local milieu of F-actin at the cell edge, is essential to capture the EGF-induced barbed-end amplification observed experimentally.     

Cofilin produces newly polymerized actin filaments that are preferred for dendritic nucleation by the Arp2/3 complex.

One of the earliest events in the process of cell motility is the massive generation of free actin barbed ends, which elongate to form filaments adjacent to the plasma membrane at the tip of the leading edge. Both cofilin and Arp2/3 complex have been proposed to contribute to barbed end formation during cell motility. Attempts to assess the functions of cofilin and Arp 2/3 complex in vivo indicate that both cofilin and Arp2/3 complex contribute to actin polymerization: cofilin by severing and Arp2/3 by nucleating and branching. In order to determine if the activities of cofilin and Arp2/3 complex interact, we employed a light microscope-based assay to visualize actin polymerization directly in the presence of both proteins. The results indicate that cofilin generates barbed ends to increase the mass of freshly polymerized F-actin but does not directly affect the activity of Arp2/3 complex. However, while ADP, ADP-Pi, and newly polymerized ATP-filaments are all capable of supporting Arp2/3-mediated branching, newly polymerized F-actin supports most of the Arp2/3-induced branch formation. The results suggest that, in vivo, cofilin contributes to barbed end formation by inducing the initial increase in the number of barbed ends leading to increased ATP-F-actin, which in turn supports higher levels of dendritic nucleation by active Arp2/3 complex.     

Rac1 and Rac2 differentially regulate actin free barbed end formation downstream of the fMLP receptor

Actin assembly at the leading edge of migrating cells depends on the availability of high-affinity free barbed ends (FBE) that drive actin filament elongation and subsequent membrane protrusion. We investigated the specific mechanisms through which the Rac1 and Rac2 small guanosine triphosphatases (GTPases) generate free barbed ends in neutrophils. Using neutrophils lacking either Rac1 or Rac2 and a neutrophil permeabilization model that maintains receptor signaling to the actin cytoskeleton, we assessed the mechanisms through which these two small GTPases mediate FBE generation downstream of the formyl-methionyl-leucyl-phenylalanine receptor. We demonstrate here that uncapping of existing barbed ends is mediated through Rac1, whereas cofilin- and ARP2/3-mediated FBE generation are regulated through Rac2. This unique combination of experimental tools has allowed us to identify the relative roles of uncapping (15%), cofilin severing (10%), and ARP2/3 de novo nucleation (75%) in FBE generation and the respective roles played by Rac1 and Rac2 in mediating actin dynamics.     

The F-actin side binding activity of the Arp2/3 complex is essential for actin nucleation and lamellipod extension

Most eukaryotic cells rely on localized actin polymerization to generate and sustain the protrusion activity necessary for cell movement [1, 2]. Such protrusions are often in the form of a flat lamellipod with a leading edge composed of a dense network of actin filaments [3, 4]. The Arp2/3 complex localizes within that network in vivo [3, 4] and nucleates actin polymerization and generates a branched network of actin filaments in vitro [5-7]. The complex has thus been proposed to generate the actin network at the leading edge of crawling cells in vivo [3, 4, 8]. However, the relative contributions of nucleation and branching to protrusive force are still unknown. We prepared antibodies to the p34 subunit of the Arp2/3 complex that selectively inhibit side binding of the complex to F-actin. We demonstrate that side binding is required for efficient nucleation and branching by the Arp2/3 complex in vitro. However, microinjection of these antibodies into cells specifically inhibits lamellipod extension without affecting the EGF-stimulated appearance of free barbed ends in situ. These results indicate that while the side binding activity of the Arp2/3 complex is required for nucleation in vitro and for protrusive force in vivo, it is not required for EGF-stimulated increases in free barbed ends in vivo. This suggests that the branching activity of the Arp2/3 complex is essential for lamellipod extension, while the generation of nucleation sites for actin polymerization is not sufficient.     

Microscopic evidence that actin-interacting protein 1 actively disassembles actin-depolymerizing factor/Cofilin-bound actin filaments

Actin-depolymerizing factor (ADF)/cofilin and gelsolin are the two major factors to enhance actin filament disassembly. Actin-interacting protein 1 (AIP1) enhances fragmentation of ADF/cofilin-bound filaments and caps the barbed ends. However, the mechanism by which AIP1 disassembles ADF/cofilin-bound filaments is not clearly understood. Here, we directly observed the effects of these proteins on filamentous actin by fluorescence microscopy and gained novel insight into the function of ADF/cofilin and AIP1. ADF/cofilin severed filaments and AIP1 strongly enhanced disassembly at nanomolar concentrations. However, gelsolin, gelsolin-actin complex, or cytochalasin D did not enhance disassembly by ADF/cofilin, suggesting that the strong activity of AIP1 cannot be explained by simple barbed end capping. Barbed end capping by ADF/cofilin and AIP1 was weak and allowed filament elongation, whereas gelsolin or gelsolin-actin complex strongly capped and inhibited elongation. These results suggest that AIP has an active role in filament severing or depolymerization and that ADF/cofilin and AIP1 are distinct from gelsolin in modulating filament elongation.     

Protein kinase D regulates cofilin activity through p21-activated kinase 4

Dynamic reorganization of the actin cytoskeleton at the leading edge is required for directed cell migration. Cofilin, a small actin-binding protein with F-actin severing activities, is a key enzyme initiating such actin remodeling processes. Cofilin activity is tightly regulated by phosphorylation and dephosphorylation events that are mediated by LIM kinase (LIMK) and the phosphatase slingshot (SSH), respectively. Protein kinase D (PKD) is a serine/threonine kinase that inhibits actin-driven directed cell migration by phosphorylation and inactivation of SSH. Here, we show that PKD can also regulate LIMK through direct phosphorylation and activation of its upstream kinase p21-activated kinase 4 (PAK4). Therefore, active PKD increases the net amount of phosphorylated inactive cofilin in cells through both pathways. The regulation of cofilin activity at multiple levels may explain the inhibitory effects of PKD on barbed end formation as well as on directed cell migration.     

Arp2/3 complex and actin depolymerizing factor/cofilin in dendritic organization and treadmilling of actin filament array in lamellipodia.

The leading edge (approximately 1 microgram) of lamellipodia in Xenopus laevis keratocytes and fibroblasts was shown to have an extensively branched organization of actin filaments, which we term the dendritic brush. Pointed ends of individual filaments were located at Y-junctions, where the Arp2/3 complex was also localized, suggesting a role of the Arp2/3 complex in branch formation. Differential depolymerization experiments suggested that the Arp2/3 complex also provided protection of pointed ends from depolymerization. Actin depolymerizing factor (ADF)/cofilin was excluded from the distal 0.4 micrometer++ of the lamellipodial network of keratocytes and in fibroblasts it was located within the depolymerization-resistant zone. These results suggest that ADF/cofilin, per se, is not sufficient for actin brush depolymerization and a regulatory step is required. Our evidence supports a dendritic nucleation model (Mullins, R.D., J.A. Heuser, and T.D. Pollard. 1998. Proc. Natl. Acad. Sci. USA. 95:6181-6186) for lamellipodial protrusion, which involves treadmilling of a branched actin array instead of treadmilling of individual filaments. In this model, Arp2/3 complex and ADF/cofilin have antagonistic activities. Arp2/3 complex is responsible for integration of nascent actin filaments into the actin network at the cell front and stabilizing pointed ends from depolymerization, while ADF/cofilin promotes filament disassembly at the rear of the brush, presumably by pointed end depolymerization after dissociation of the Arp2/3 complex.     

Efficient Salmonella entry requires activity cycles of host ADF and cofilin

Entry of Salmonella into mammalian cells is strictly dependent on the reorganization of actin cytoskeleton induced by a panel of Salmonella type III secreted proteins. Although several factors have been identified to be responsible for inducing the actin polymerization and stability, little is known about how the actin depolymerization contributes to Salmonella-induced actin rearrangements. We report here that activity cycles of host actin depolymerizing factor (ADF and cofilin) are modulated by Salmonella during bacterial entry. Efficient Salmonella internalization involves an initial dephosphorylation of ADF and cofilin followed by phosphorylation, suggesting that ADF and cofilin activities are increased briefly. Expression of a kinase dead form of an ADF/cofilin kinase (LIM kinase 1) or a catalytically inactive ADF/cofilin phosphatase (Slingshot), but not constitutively active LIM kinase 1 or wild-type Slingshot, resulted in decreased invasion. These data suggest that ADF/cofilin activities play a key role in the actin polymerization/depolymerization process induced by Salmonella. The activation of ADF/cofilin is brief and has to be reversed to facilitate efficient bacterial entry. Surprisingly, co-expression of constitutive active ADF and cofilin prevented efficient Salmonella entry, whereas expression of either one alone had no effect. We propose that ADF and cofilin actin-dynamizing activities and their activity cycling via phosphorylation are required for efficient Salmonella internalization.     

Cofilin/ADF is required for cell motility during Drosophila ovary development and oogenesis

The driving force behind cell motility is the actin cytoskeleton. Filopodia and lamellipodia are formed by the polymerization and extension of actin filaments towards the cell membrane. This polymerization at the barbed end of the filament is balanced by depolymerization at the pointed end, recycling the actin in a 'treadmilling' process. One protein involved in this process is cofilin/actin-depolymerizing factor (ADF), which can depolymerize actin filaments, allowing treadmilling to occur at an accelerated rate. Cofilin/ADF is an actin-binding protein that is required for actin-filament disassembly, cytokinesis and the organization of muscle actin filaments. There is also evidence that cofilin/ADF enhances cell motility, although a direct requirement in vivo has not yet been shown. Here we show that Drosophila cofilin/ADF, which is encoded by the twinstar (tsr) gene, promotes cell movements during ovary development and oogenesis. During larval development, cofilin/ADF is required for the cell rearrangement needed for formation of terminal filaments, stacks of somatic cells that are important for the initiation of ovarioles. It is also required for the migration of border cells during oogenesis. These results show that cofilin/ADF is an important regulator of actin-based cell motility during Drosophila development.     

N-terminal domains of the class ia phosphoinositide 3-kinase regulatory subunit play a role in cytoskeletal but not mitogenic signaling

Phosphoinositide (PI) 3-kinases are required for the acute regulation of the cytoskeleton by growth factors. We have shown previously that in the MTLn3 rat adenocarcinoma cells line, the p85/p110alpha PI 3-kinase is required for epidermal growth factor (EGF)-stimulated lamellipod extension and formation of new actin barbed ends at the leading edge of the cell. We have now examined the role of the p85alpha regulatory subunit in greater detail. Microinjection of recombinant p85alpha into MTLn3 cells blocked both EGF-stimulated mitogenic signaling and lamellipod extension. In contrast, a truncated p85(1-333), which lacks the SH2 and iSH2 domains and does not bind p110, had no effect on EGF-stimulated mitogenesis but still blocked EGF-stimulated lamellipod extension. Additional deletional analysis showed that the SH3 domain was not required for inhibition of lamellipod extension, as a construct containing only the proline-rich and breakpoint cluster region (BCR) homology domains was sufficient for inhibition. Although the BCR domain of p85 binds Rac, the effects of the p85 constructs were not because of a general inhibition of Rac signaling, because sorbitol-induced JNK activation in MTLn3 cells was not inhibited. These data show that the proline-rich and BCR homology domains of p85 are involved in the coupling of p85/p110 PI 3-kinases to regulation of the actin cytoskeleton. These data provide evidence of a distinct cellular function for the N-terminal domains of p85.     

Spatial and temporal control of cofilin activity is required for directional sensing during chemotaxis

BACKGROUND: Previous work has led to the hypothesis that cofilin severing, as regulated by PLC, is involved in chemotactic sensing. We have tested this hypothesis by investigating whether activation of endogenous cofilin is spatially and temporally linked to sensing an EGF point source in carcinoma cells. RESULTS: We demonstrate that inhibition of endogenous cofilin activity with either siRNA or overexpression of LIMK suppresses directional sensing in carcinoma cells. LIMK siRNA knockdown, which suppresses cofilin phosphorylation, and microinjection of S3C cofilin, a cofilin mutant that is constitutively active and not phosphorylated by LIMK, also inhibits directional sensing and chemotaxis. These results indicate that phosphorylation of cofilin by LIMK, in addition to cofilin activity, is required for chemotaxis. Cofilin activity concentrates rapidly at the newly formed leading edge facing the gradient, whereas cofilin phosphorylation increases throughout the cell. Quantification of these results indicates that the amplification of asymmetric actin polymerization required for protrusion toward the EGF gradient occurs at the level of cofilin but not at the level of PLC activation by EGFR. CONCLUSIONS: These results indicate that local activation of cofilin by PLC and its global inactivation by LIMK phosphorylation combine to generate the local asymmetry of actin polymerization required for chemotaxis.     

Reactive oxygen species regulate protrusion efficiency by controlling actin dynamics

Productive protrusions allowing motile cells to sense and migrate toward a chemotactic gradient of reactive oxygen species (ROS) require a tight control of the actin cytoskeleton. However, the mechanisms of how ROS affect cell protrusion and actin dynamics are not well elucidated yet. We show here that ROS induce the formation of a persistent protrusion. In migrating epithelial cells, protrusion of the leading edge requires the precise regulation of the lamellipodium and lamella F-actin networks. Using fluorescent speckle microscopy, we showed that, upon ROS stimulation, the F-actin retrograde flow is enhanced in the lamellipodium. This event coincides with an increase of cofilin activity, free barbed ends formation, Arp2/3 recruitment, and ERK activity at the cell edge. In addition, we observed an acceleration of the F-actin flow in the lamella of ROS-stimulated cells, which correlates with an enhancement of the cell contractility. Thus, this study demonstrates that ROS modulate both the lamellipodium and the lamella networks to control protrusion efficiency.     

Identification of functional residues on Caenorhabditis elegans actin-interacting protein 1 (UNC-78) for disassembly of actin depolymerizing factor/cofilin-bound actin filaments

Actin-interacting protein 1 (AIP1) is a WD40 repeat protein that enhances actin filament disassembly in the presence of actin-depolymerizing factor (ADF)/cofilin. AIP1 also caps the barbed end of ADF/cofilin-bound actin filament. However, the mechanism by which AIP1 interacts with ADF/cofilin and actin is not clearly understood. We determined the crystal structure of Caenorhabditis elegans AIP1 (UNC-78), which revealed 14 WD40 modules arranged in two seven-bladed beta-propeller domains. The structure allowed for the mapping of conserved surface residues, and mutagenesis studies identified five residues that affected the ADF/cofilin-dependent actin filament disassembly activity. Mutations of these residues, which reside in blades 3 and 4 in the N-terminal propeller domain, had significant effects on the disassembly activity but did not alter the barbed end capping activity. These data support a model in which this conserved surface of AIP1 plays a direct role in enhancing fragmentation/depolymerization of ADF/cofilin-bound actin filaments but not in barbed end capping.